Abstract

Gugulipid (GL), extract of Indian Ayurvedic medicinal plant Commiphora mukul, has been used to treat a variety of ailments. We report an anticancer effect and mechanism of GL against human prostate cancer cells. Treatment with GL significantly inhibited the viability of human prostate cancer cell line LNCaP (androgen-dependent) and its androgen-independent variant (C81) with an IC(50) of ∼1 μM (24-h treatment), at pharmacologically relevant concentrations standardized to its major active constituent z-guggulsterone. The GL-induced growth inhibition correlated with apoptosis induction as evidenced by an increase in cytoplasmic histone-associated DNA fragmentation and sub-G(0)/G(1)-DNA fraction, and cleavage of poly(ADP-ribose) polymerase. The GL-induced apoptosis was associated with reactive oxygen species (ROS) production and c-Jun NH(2)-terminal kinase (JNK) activation. The induction of proapoptotic Bcl-2 family proteins Bax and Bak and a decrease of antiapoptotic Bcl-2 protein Bcl-2 were observed in GL-treated cells. SV40 immortalized mouse embryonic fibroblasts derived from Bax-Bak double-knockout mice were significantly more resistant to GL-induced cell killing compared with wild-type cells. It is interesting to note that a representative normal prostate epithelial cell line (PrEC) was relatively more resistant to GL-mediated cellular responses compared with prostate cancer cells. The GL treatment caused the activation of JNK that functioned upstream of Bax activation in apoptosis response. The GL-induced conformational change of Bax and apoptosis were significantly suppressed by genetic suppression of JNK activation. In conclusion, the present study indicates that ROS-dependent apoptosis by GL is regulated by JNK signaling axis.

Effect of GL [GL contains ∼3.75% z-Gug and was standardized to z-Gug (micromoles); A, B, and D] and z-Gug (C) on survival of LNCaP, C81, and PrEC cells determined by the colonogenic assay (A) and trypan blue dye exclusion assay (B–D). Cells were treated with different concentrations of GL or z-Gug for 24 h. Columns, mean of three determinations; bars, S.E. *, significantly different (P < 0.05) compared with DMSO-treated control by one-way ANOVA followed by Dunnett's test. Similar results were observed in two independent experiments. Representative data from a single experiment are shown.

GL induced apoptosis in LNCaP and C81 cells but not in normal human prostate epithelial cells PrEC, determined by quantitation of cytoplasmic histone associated DNA fragmentation (A), flow cytometry analysis of sub-G0/G1 cell phase (B), and immunoblotting cleavage of PARP (C). Cells were treated with the indicated concentrations of GL or DMSO (control) for 24 h. Results in A and B are expressed as enrichment factor relative to cells treated with DMSO (control). Results are mean ± S.E. (n = 3). *, significantly different (P < 0.05) between the indicated groups by one-way ANOVA followed by Dunnett's test. In C, the cleaved PARP by immunoblotting using lysates from GL-treated or DMSO-treated LNCaP and C81 cells. The blot was stripped and reprobed with anti-α-tubulin antibody to ensure equal protein loading. Similar results were observed in at least two independent experiments. Representative data from a single experiment are shown.

GL-induced ROS production was involved in apoptotic cell death caused by GL. GL caused ROS generation in C81 (A) and LNCaP (B) cells in dose- (A, left, for C81) and time- (A, right, for C81 and B for LNCaP) dependent manner but not in PrEC (C). D, NAC protected against GL-mediated ROS production and apoptosis. LNCaP cells were treated with 10 mM NAC for 2 h and then exposed to 5 μM GL standardized to z-Gug for 30 min (D, left) or 24 h (D, right). In A to D, results are mean ± S.E. (n = 3). *, significantly different (P < 0.05) between the indicated groups by one-way ANOVA followed by Dunnett's test (A and C, left) and Bonferroni multiple comparison test (D) and by paired t test (A and B, right). Experiments were repeated twice with triplicate measurements in each experiment. The results were consistent, and representative data from a single experiment are shown.

A, immunoblotting for Bax, Bak, and Bcl-2 proteins using lysates from LNCaP cells treated with DMSO (control) or 5 μM GL standardized to z-Gug for the indicated time periods. B, immunoblotting for Bax protein using lysates from PrEC treated with DMSO (control) or 5 μM GL standardized to z-Gug for the indicated time periods. C, immunoblotting for cleaved PARP protein from the SV40-immortalized mouse embryonic fibroblasts derived WT and Bax and Bak double-knockout mice treated with DMSO (control) or 2.5 and 5 μM GL standardized to z-Gug for 24 h. For A to C, the blots were stripped and reprobed with antiactin antibody to normalize for differences in protein level. The numbers on top of the immunoreactive bands represent a change in protein levels relative to DMSO-treated cells. Immunoblotting for each protein was performed at least twice using independently prepared lysates. The cell survival (D) and cytoplasmic histone-associated DNA fragmentation (E) in LNCaP cells treated with DMSO (control) or 2.5 and 5 μM GL standardized to z-Gug for 24 h. For D and E, columns, mean (n = 3); bars, S.E. *, significantly different (P < 0.05) compared with corresponding DMSO-treated control by one-way ANOVA followed by Dunnett's test. Each experiment was performed at least twice with triplicate measurements in each experiment. The results were consistent and representative data from a single experiment are shown.

The GL treatment increased activating phosphorylation of JNK in LNCaP cells, but not in PrEC. Immunoblotting for phospho-JNK, phosphor-Erk, and phospho-p38 MAPK using lysates from LNCaP cells (A) and immunoblotting for phospho-JNK using lysates from PrEC treated with DMSO (control) or 5 μM GL standardized to z-Gug or 5 μM z-Gug for the indicated time periods (B). The blots were stripped and reprobed with anti-actin antibody to ensure equal protein loading. Immunoblotting for each protein was performed twice using independently prepared lysates and the results were similar. Representative data from a single experiment are shown. Fold change in phospho/total protein level relative to DMSO-treated control at each time point is shown on top of the immunoreactive band. C, NAC protected against GL-mediated JNK activation. LNCaP cells were treated with 10 mM NAC for 2 h and then with or without 2.5 μM GL standardized to z-Gug for 8 h. The cellular lysates from these groups were performed for immunoblotting of phospho-c-Jun. The blots were stripped and reprobed with anti-actin antibody to ensure equal protein loading. The numbers on top of the immunoreactive bands represent the change in protein levels relative to corresponding DMSO-treated control. Immunoblotting for the protein was performed twice using independently prepared lysates and the results were similar. Representative data from a single experiment are shown.